Polyethylene glycols are a family of polymers produced from the condensation of ethylene glycol, usually initiated with base, and have the general formula, HO—(CH2CH2O)n—H, where n, the number of ethylene glycol groups, is greater than or equal to 4. Generally, the designation of a polyethylene glycol (PEG) includes a number that corresponds to its average molecular weight, Mn, which indicates the extent of polydispersity. All commerical PEG's are polydisperse. For example, polyethylene glycol 1500 refers to a mixture of polyethylene glycols having an average value n of between 19 and 48 (with some even smaller and some larger) and a molecular weight range from about 800 to 2100 grams/mole.
The properties of polyethylene glycols vary with the polymer's molecular weight. Polyethylene glycols have been used in plasticizers, softeners and humectants, ointments, polishes, paper coatings, mold lubricants, bases for cosmetics and pharmaceuticals, solvents, binders, metal and rubber processing, permissible additives to foods and animal feed, and laboratory reagents, among others. Polyethylene glycols generally are linear or branched. PEGs are neutral polyether molecules that are soluble in water and organic solvents. In addition to the uses noted above, polyethylene glycols have proven valuable in many biotechnical and biomedical applications. Polyethylene glycols have been advantageously employed in these applications for their ability to impart water solubilization and surface protective properties, and also because these polymers are only weakly immunogenic.
Polyethylene glycols also have been covalently coupled to proteins to alter their properties in ways that extend their potential uses. Due to in vivo instability, the efficacy of a number of theapeutic proteins is severely limited. While many approaches to stablization of such proteins have been made, the covalent modification of proteins with hydrophilic polymers, such as dextran and polyethylene glycols, has been most successful. Typically, polyethylene glycol-protein conjugates are more stable than the native protein in vivo and often, the modified proteins exhibit enhanced resistance to proteolytic degradation. The result is an increase in the therapeutic proteins' life in circulation and a reduction in its immunogneicity. In some instances, the therapeutic efficacy of these conjugates is greatly enhanced compared to the native protein.
The improved performance of PEG-modified conjugates has resulted in their development as therapeutic agents. Examples of polyethylene glycol-modified proteins include PEG-adenosine deaminase (PEG-ADA), which has been used in enzyme replacement therapy for immunodeficiency due to ADA deficiency (M. S. Hershfield, Clin. Immunol. Immuno. Pathol., Vol. 76, S 228-232, 1995); PEG-recombinant human granulocyte colony stimulating factor (PEG-rhG-CSF), which showed an increase in stability and retention of in vivo bioactivity and has been suggested as a suitable form of the protein for inclusion in an oral delivery formulation (P. K. E. Jensen et al., Pharm. Res., Vol. 13, pp. 102-107, 1996); PEG-natural human tumor necrosis factor alpha, which showed a gradual decrease in specific activity with increasing degree of PEG-modification and a drastic increase in plasma half-life upon PEG-modification (Y. Tsutsumi et al., Br. J. Cancer, Volume 71, pp. 963-968, 1995); PEG-recombinant human interleukin-2, which retains the in vitro and in vivo activity of interleukin-2, but exhibits a markedly prolonged circulating half-life (T. Menzel et al., Cancer Bio. Ther., Vol. 8, pp. 199-212, 1993); and PEG-asparaginase, which has shown promise in patients suffering from acute lymphocytic leukemia (N. Burnham, Am. J. Hosp. Pharm., Vol. 52, pp. 210-218, 1994). Polyethylene glycol conjugates of oligonucleotides also have been prepared and show a more than tenfold increase in exonuclease stability (A. Jaschke et al., Nucleic Acids Research, Vol. 22, pp. 4810-4817, 1994).
Other PEG-modified proteins include, inter alia, papain (C. Woghiren et al., Bioconjugate Chemistry, Vol. 4, pp. 314-318, 1993), asialofetuin (L. Roseng et al., J. Biol. Chem., Vol. 267, pp. 22987-22993, 1992), collagen (C. J. Doillon et al., Biomaterial Sciences Polymers, Vol. 6, pp. 715-728, 1994), RGDT peptides (I. Saiki, Japanese J. Cancer Research, Vol. 84, pp. 558-565, 1993), serum IgG (R. Cunningham et al., J. Immunol. Methods, Vol. 152, pp. 177-190, 1992), alpha 1-proteinase inhibitor (A. Mast et al., J. Lab. Clin. Med., Vol. 116, pp. 58-65, 1990), growth hormone releasing factor (A. Felix, Int. J. Peptide Protein Research, Vol. 46, pp. 253-264, 1995), basic fibroblast growth factor (S. Kusstatscher et al., J. Pharmacol. Exp. Ther., Vol. 275, pp. 456-61, 1995), and catalase, uricase, honey bee venom, hemoglobin, and ragweed pollen extract. As indicated by the number of utilities noted above, polyethylene glycol has recently been widely used to develop new therapeutic agents. Two of the best general references to these applications are the monographs edited by J. Milton Harris:                (a) Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, J. Milton Harris, ed., Plenum Press, New York, 1992.        (b) Poly(ethylene glycol): Chemistry and Biological Applications, ACS Symposium Series, Vol. 680, J. Milton Harris and Samuel Zalipsky, eds., American Chemical Society, Washington, D.C., 1997.        
Despite the widespread use of polyethylene glycols to modify therapeutic agents, their use has not been without associated disadvantages. The covalent attachment of polyethylene glycol to superoxide dismutase produces a heterogeneous mixture of modified protein species. The heterogeneity of the product derives from, in part the polydispersity of the polyethylene glycol reagent (J. Snyder, et al., J Chromatography, Vol. 599, pp. 141-155, 1992.)
These associated disadvantages are due solely to the polydisperse and multicomponent nature of the polymeric polyethylene glycols which are currently being used exclusively, as no discrete alternatives are available. Even the polydispersed products available and being applied are limited to molecular weights above about 2000 (average n greater than about 40-50) due to the higher Mn and molecular weight distributions of the lower MW's, among others. Comments in a recent article, “Synthesis of Polyamide Oligomers Based on 14-Amino-3,6,9,12-tetraoxatetradecanoic Acid,” S. M. Ansell, et al., Bioconjugate Chem., 11, 14-21 (2000), are indicative of the extent of the real problems, as well as pointing directly to the value and importance of the dPEG's.
Fine-tuning the behavior of these systems presents considerable challenges, in part due to the nature of the PEGs that are commercially available. Systems being developing for drug delivery are based on liposomes or lipid-based particulates for the delivery of conventional therapeutic agents or genetic medicines. Problems include a limited selection of molecular weights for both monofunctional and heterobifunctional derivatives, polydispersity variablility, and average molecular weight variability of these compounds, which potentially leads to reproducibility issues with different batches and varible exchange rates associates with different size populations. In addition, the presence of low molecular weight homomers in PEG would result in a small population of PEG-lipid, which would not be rapidly exchanged out of formulations in vivo and potentially could have major implications in systems where immune responses against PEG are an issue.
Commercially available polyethylene glycols having molecular weights greater than about 250 grams/mole are available only as mixtures of varying length polymers. The range of PEG polymer lengths results form the polymerization process by which the PEG polymers are prepared. Commercially available PEG polymers include polymers, inter alia, having average molecular weights of 200, 300, 400, 600, 900, 1000, 1500, 2000, 3400, 4000, 4600, 8000, 10000, 20000, 35000, 200000, 300000, 400000, 600000, 900000, 1000000, 2000000, 4000000, 5000000, 7000000, 8000000, etc. The exact composition of these mixtures is never provided and is of generally a broad range of MW range per the example shown in FIG. 1. However, where terminal monomethyl ethers are desired, these MW ranges are considered to be less broad or narrower then the bis-hydroxyl. This is consistent with the way they are polymerized and initiated with the methoxide, therefore growing randomly from only one end versus growing at both ends of the polymer. Statistically the methoxy terminated would have a narrower range of MWs. However, due to the presence of water in these initiations, there is as much as 25% of the polydiol present, resulting in an even broader MW range.
Accordingly, there remains a need in the art for alternatives to PEG polymers composed of a mixture of lengths and molecular weights to overcome the difficulties associated with the preparation, process variability and/or reproducibility, purification, characterization, and therapeutic administration of such PEG mixtures. The present invention seeks to fulfill these needs and provides further related advantages.
A number of methods have been tried in literature using a more conventional organic synthetic approach to making discrete polyethylene glycol oligomers. Booth and co-workers tried using a convergent-like approach. They did not place protecting groups on the ends of the diols and obtained complex mixtures of oligomers. These could only be separated using complicated and time consuming methods in very low yields. The reaction times also were very long (Refs.: A. Marshall, R. H. Mobbs and C. Booth, “Preparation of Ethylene Glycol Oligomers,” European Polymer Journal, Vol. 16, pp. 881 to 885, 1980; H. H. Teo, R. H. Mobbs and C. Booth, “Preparation of Ethylene Glycol Oligomers-II,” European Polymer Journal, Vol. 18, pp. 541 to 544, 1982; S. G. Yeates, H. H. Teo, R. H. Mobbs and C. Booth, “Ethylene Glycol Oligomers,” Makromol. Chem., Vol. 185, pp. 1559 to 1563, 1984).
Harris demonstrated a solid phase synthetic approach to making monodispersed oligomers. (Ref. J. Milton Harris, et al., Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, J. Milton Harris, ed., Plenum Press, New York, 1992, pp. 371-381). However, this is not a practical approach for scale-up and he found that due to the nature of using a polymer support, the steps are very slow and require high temperatures to complete. And as is the case with polymer support reactions, the valuable building blocks have to be used in considerable excess to achieve complete reactions. Dr. Harris also mentions that their attempts at solution phase synthesis “have proven to produce low yields of products that are difficult to purify.”
Svendhem, et al., have made a series of monodispersed amino-PEG-alcohols using ether coupling chemistry similar to the stepwise approach, but they found that “the couplings were sluggish and slow, and purification by HPLC was necessary to obtain pure compounds (on mg scales). Efforts to optimize the yields by changing solvent and bases, or phase-transfer conditions were all unsuccessful.” (Ref. S. Svedhem, C.-A. Hollander, J. Shi, P. Konradsson, B. Liedberg, and S. C. T. Svensson, “Synthesis of a Series of Oligo(ethylene glycol)-Terminated Alkanethiol Amides Designed to Address Structure and Stability of Biosensing Interfaces,” J. Org. Chem., 66, 4494-4503 (2001)).
More recently Chen and Baker have demonstrated a very limited example of the convergent synthesis of monodispersed PEGs. However, the conditions for the only protecting group are extremely severe, the reaction times long, and they show only examples using small commercially available diols. (Ref. “Synthesis and Properties of ABA Amphiphiles,” Yiyan Chen and Gregory L. Baker, J. Org. Chem., 64, 6870-6873 (1999).TrO—(CH2CH2O)a—H+TsO—(CH2CH2O)b-Ts→TrO—(CH2CH2O)2a+b-Tr→HO—(CH2CH2O)2a+b—H(H2, Pd/C, 50 atm, 48 h) 2a+b≦14
In addition to the other limitations, each of these methods relies on having a preformed or pregenerated alkoxide species, which is very basic and nucleophilic, and has the potential of leading to varying amounts of undesirable by-products, either via side reactions of with secondary reactions on the desired product. A preferred embodiment of the current invention significantly reduces and maybe eliminate this potential by generating the reactive species in situ.
More recently in published application U.S. 2003/0004304 A1 (Ekwuribe, et al., “Methods of Synthesizing Substantially Monodispersed Mixtures of Polymers having Polyethyene Glycol Moieties,” publication date, Jan. 2, 2003), the inventors propose a stepwise-like approach to make “substantially mondispersed mixture of polymers.” This patent application proposes a process for making “substantially monodispersed mixtures of polymers” using a process directed to their interests in synthesizing the smaller versions of their “substantially monodispersed mixtures of polymers” attached to highly lipophilic substituents. Ekwuribe still speaks in terms of “mixtures” of polymers, rather than discrete species, as in the present invention. Processing details also are quite different. Production of discrete PEGs with a predetermined number of ethylene oxide units is not possible. Moreover, Applicants' stepwise synthesis scheme is totally absent from Ekwuribe.